New T08-005A Allelic Discrimination Analysis - Cole-Parmer · 2018. 7. 1. · T08-005A: Allelic...

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Technical Note T08-005A

Allelic Discrimination Analysis

General Introduction to Data Analysis The aim of this Technical Note is to explain the principle of allelic discrimination analysis and to guide you through

the experimental set up and analysis of data. It begins with a general introduction to data analysis.

Results Editor

The starting point for data analysis is the Results Editor. Before an analysis method has been set, the Results Editor

home screen will display the plate layout and thermal cycling program. Each stage where readings have been made

will have its own tab showing a graph of raw data.

Selecting the Analysis Method

The Analysis Selection box on the Results Editor home screen allows the user to define the method of analysis to

be applied to the readings gathered during the PCR run. Highlighting a stage name and pressing the Edit button will

launch the Analysis Wizard Selection screen and allow an Analysis Method to be assigned for that stage.

The Analysis Wizards

Once an analysis method has been defined, a series of default settings will automatically analyse the data. The

defaults can be viewed in the Analysis Wizards and edited if required. The intuitive Analysis Wizards explain in

detail the mathematics behind the analysis settings and will lead you through each stage of the analysis setup.

With experience, analysis can be quickly performed using the Analysis Properties displayed in the Parameters (PAR)

box feature found on each data graph.

Figure 1: The Results Editor home screen.

Each stage where readings have been made will have a separate results

tab enabling analysis of the data for that particular stage.

The Analysis Selection box displays the stage name as assigned in the

program setup. Only those stages that have been assigned with reads are

displayed since stages without reads have no data to analyse. Double-click

on a stage name, or highlight a stage name then click on Edit to launch the

Analysis Wizard Selection screen.

Figure 2: The Analysis Wizard Selection screen.

Analysis Method: The drop-down menu lists analysis types appropriate to

the selected stage. These will be dependent on the number of reads and

the number of cycles programmed into the run.

Dye name: The name of the dyes selected in the program setup will be

displayed.

Dye Usage: Assign a Dye Usage from the list in the drop-down menu. There

will be one dye usage box for each read present in the stage.

Cancel: Aborts the procedure and takes the user back to the Results Editor.

Finish: Accepts all the default analysis settings for the analysis method

chosen and closes the Wizard.

Reset defaults: Returns all analysis parameter settings to the defaults.

Back/Next: Allows the user to move between screens in the Analysis

Wizards.

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T08-005A: Allelic Discrimination Analysis

Analysis Method Options

The following analysis options are available in Quansoft:

Baseline This simple analysis method allows for correction of differences in background fluorescence. It is also incorporated

into many of the other analysis methods.

Quantification

Quantification analysis is used to determine the absolute or relative quantity of a target DNA template in a given

test sample by measuring the cycle-to-cycle change in the fluorescent signal. The fluorescent signal increases

proportionately to the amount of amplified DNA and quantification is performed either by comparison of the

fluorescence of a PCR product of unknown concentration with that of several dilutions of an external standard, or

by comparing the fluorescence of one product relative to another. To be able to make this comparison, the

fluorophore is measured at a point in the amplification where the reaction efficiency can be considered optimal.

This is generally around the cycle at which an increase in fluorescence is first detected.

Dissociation curve

Dissociation curve analysis can add to the information obtained from the PCR. Also known as melting curve

analysis, it measures the temperature at which the DNA strands separate into single strands. This provides a

measurement of the melting temperature or Tm, taken as the point at which 50% of the double stranded DNA

(dsDNA) molecules are dissociated. Using the easy-to-program ‘ramp’ function, PrimeQ will perform a thermal

ramping program that can be used to determine the Tm of the PCR product. This analysis provides the user with

extra confidence in experiments using intercalating dye chemistry for identifying amplification of non-specific

products or contamination.

Plus-minus scoring

This analysis exploits PrimeQ’s fluorescence technology to determine with ease and accuracy the presence or

absence of a PCR product in any given sample. Input data can either be kinetic (where readings are taken

throughout the amplification stage) or end-point (readings taken at the end of the run). The software scores the

samples as positive or negative according to user-defined thresholds.

Allelic discrimination

Users of PrimeQ have the option of this powerful technique capable of detecting single nucleotide differences

(SNPs). It can be used to discriminate between genotypes, mutations and polymorphisms within or between

samples simply by comparing the fluorescence signal obtained using allele-specific, dye-labeled probes.

Multi-read

This is a simple end point analysis method which will report the average fluorescence of a selected number of

readings. It is useful for assays other than PCR, for example fluorescence-based DNA assays, where just the

fluorescence of a sample needs to be measured; in this way PrimeQ can be used as a simple fluorescence plate

reader or fluorimeter.



Analysis method: Allelic Discrimination Allelic discrimination is a technique that is able to detect single base pair differences. It is used to discriminate

between genotypes, mutations and polymorphisms within or between samples by comparing the fluorescence

signal obtained using allele-specific, dye-labelled probes. It is a qualitative rather than a quantitative analysis

method; the input data for the analysis is usually the end-point fluorescence (a few readings taken at the end of

the run – at least two are required).

Principle

A single base mismatch within the region complementary to the probe will reduce its stability and therefore the

Tm. The most common technique to use is the hydrolysis probe assay using dual-labelled probes. A multiplex assay

must be run for this type of analysis as two probes, labelled with different reporter dyes are required. One probe

must be 100% specific for one allele and the second probe 100% specific for the mutant allele. During the PCR, the

fluorescent probes anneal specifically to complementary sequences between the forward and reverse primers sites

on the template DNA. During the extension cycle of the PCR, Taq DNA polymerase cleaves the hybridized probe

and due to separation of the reporter dye from the quenchers, an increase in fluorescence is seen.

Thermodynamically it is far more favourable for only the matching probe to bind to the template than the probe

containing the mismatch.

Setting up and running the experiment

If the entire run is to be performed on PrimeQ, set up the thermal cycling profile in the usual way. You may or may

not wish to collect fluorescence data at each cycle to view the progress of the amplification; however you will need

to use fluorescence data from a few cycles at the end of the run for data analysis. This may be the last few cycles of

the amplification stage, or a separate stage may be programmed for this purpose. Examples of programs are given

in Figure 4 below.

Figure 3: Principle of allelic discrimination

analysis.

Each homozygous genotype will be

detected by a specific probe and show an

increase in fluorescence for that particular

reporter. A heterozygous genotype will

show increased fluorescence with both

reporters. A scatter plot of end point

fluorescence is used to classify and identify

the genotypes.



Allelic Discrimination Analysis Wizard setup

Once the PCR has completed, open the results file and from the Results Editor home screen set up the allelic

discrimination analysis as described below.

• In the Analysis Selection box, highlight the stage name for analysis to be applied and click Edit. The Analysis

Wizard will launch.

• Select Allelic Discrimination from the drop-down menu in the Analysis Method selection box.

• Assign a dye usage for each of the reads (e.g. Allele 1, Allele 2 etc.) and click Next. The Allelic Discrimination

Analysis Wizard will launch.

• If a passive reference dye (PRD) was assigned in the dye usage menu, the next screen will offer the option of

PRD correction. Click Next.

.

Baseline Correction

Baseline correction is only appropriate if fluorescence data has been collected throughout the amplification stage.

In this case, follow the directions in the Operator’s Manual in section 4.5. If a separate stage has been programmed

for collecting the fluorescence data for allelic discrimination analysis, set baseline correction to None then click on

Next to progress to the next screen.

Figure 4: Example programs for Allelic discrimination

assays.

(A) A standard two-step program with reads for both probes

throughout the amplification stage. Analysis would be

performed on the data from the final few cycles.

(B) As A, but with an additional stage at the end of the

amplification stage with a few reads for the allelic

discrimination analysis.

(C) No readings are taken during amplification but an

additional stage is included at the end with a few reads for

the allelic discrimination analysis.

Figure 5: Analysis Wizard Selection.

Select the Allelic Discrimination analysis method and assign dye usages.



Allelic Discrimination Analysis Method

The next screen allows you to choose which readings are to be used for allele scoring. Select the appropriate data

points then click Next.

Reporter Selection

Select which alleles to compare. The fluorescence of the first allele is plotted against that of the second and

displayed on a scatter graph. These can be swapped while viewing the results by accessing the Analysis Properties

via the PAR button on the results graph.

Report Options and Summary

The Report Options screen allows you to select data to appear in a printable report. The selection can be changed

from the Report tab in the Results Editor if necessary. The Summary screen provides a quick checklist of the

parameters set up in the Analysis Wizard.

• Click Back to change any settings or Cancel to abort the procedure.

• Click Finish to complete the set up.

Viewing the analysed results

Click on the results tab for the stage which has been set up for allelic discrimination analysis. The analysed data will

be displayed as a scatter graph similar to that in Figure 8 below. Individual graphs can be closed or enlarged for

Figure 6: Analysis Method.

Select the appropriate data points for analysis:

End-point (default): Uses the last reading only (we recommend that >1

reading is used for accuracy).

All readings: Averages all readings in the stage.

Last readings: Averages a user-specified number of last readings (the

default is 5 or 1 if there are less than 5 readings).

Specify range: Specify a range of readings to be averaged that best suit the

data (the default is the last reading).

Figure 7: Reporter Selection.

The choice of allele can also be changed when viewing the results using the

Analysis properties.



easier viewing. Clicking an individual well or a selection of wells in the plate layout will highlight just the selected

well(s) on the graphs. Clicking the Show All Wells button will re-select all wells.

The Allelic Discrimination Scatter Graph

Particular to the allelic discrimination method is the display of a scatter plot representing the fluorescence of one

allele plotted against the other. The software plots the results of the run on a scatter plot of allele 1 vs. allele 2 with

each well of the 96-well plate represented by a point on the graph.

Homozygotes will show increased fluorescence along one axis of the plot (depending on which reporter they were

detected by), while the heterozygotes appear towards the centre of the plot as they contain copies of both alleles

and therefore produce fluorescence with both dyes. Samples that do not cluster tightly may contain rare sequence

variations, sequence duplications or the PCR may not have been optimal.

Assigning Genotypes

You can define clusters of points within the plot that represent genotypic segregation within the samples; the

scoring of each sample is then presented in the Results table.

• Click Type Color in the Display box.

• Select one of the coloured Type tiles in the Type box.

Figure 8: The layout of data in the Results Editor screen for allelic

discrimination analysis.

Figure 9: The allelic discrimination scatter graph.

Four distinct clusters should be seen representing the no template

control (NTC) and three possible genotypes: homozygous A (AA), or B

(BB) and heterozygous (AB). To swap the axes or select different alleles

to compare, click the PAR button on the top right hand corner of the

scatter plot and change the Reporter Selection.



• To assign a sample to that type, either click on individual sample points or use the mouse to draw a box around

the cluster. To enhance visibility, maximize the scatter graph by clicking on the zoom icon in the top-right corner

of the pane.

The selected data points will change colour to that of the selected Type tile.

• Repeat for the remaining data until all genotypes have been defined.

Each type can be given a name which will then appear in the results table.

• Click on Change name in the Type box and an Allele Type box will open.

• Type in the required names and click OK to accept.

The Results Table

For each well the fluorescence value for each reporter dye is displayed in the Results table together with its allele

type determined from the typing assigned in the scatter graph. To change the order of the axes, enter the Analysis

Parameters using the PAR button next to the allelic discrimination scatter graph. In Reporter selection change the

order of alleles to compare.

Figure 10: Allelic discrimination graph.

Assign individual samples to a type.

Figure 11: Allele type naming.

Select names to classify each type. The names given will appear in the results table.

Figure 12: The Results Table.

The types assigned to each sample are recorded in the

results table.



Viewing and Changing the Parameters

The analysis parameters as set up in the allelic discrimination Wizard can be changed at any time and can be easily

accessed via the PAR box on the allelic discrimination graph.

PrimeQ Report

If a printed report of the analysed data is required, open the Report tab of the Results Editor to view the Plus-

Minus Scoring analysis report. To change any of the report contents, click on the Report Options icon to open up

the Report Options box. Tabs will display the report options relevant for each stage. Change as appropriate and

click Done to finish. The report can be printed or saved as a PDF file for future reference.

Saving

Saving will overwrite any previous analysis, therefore to preserve a particular analysis set up, click on File followed

by Save As… to save as a different file name.

Trademarks

FAM ™ and ROX™ are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

SYBR® is a registered trademark of Life Technologies Corporation.

Yakima Yellow® is a registered trademark of Epoch Biosciences, Inc.

Figure 13: Analysis Properties.

Click the PAR button next to the scatter graph to bring up the analysis properties for

allelic discrimination. If any settings are changed, the data will be recalculated and the

graphs and results table updated accordingly.

New T08-005A Allelic Discrimination Analysis - Cole-Parmer · 2018. 7. 1. · T08-005A: Allelic...

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